Improving blast resistance in italian rice variety baldo via crispr-cas9 editing of pi21

Overview

Rice blast, caused by the fungus Pyricularia oryzae, is one of the most damaging diseases of cultivated rice worldwide, leading to significant yield losses. This research targets the Pi21 quantitative trait locus (QTL), which is known to contribute to partial, quantitative resistance to rice blast. The project aims to apply CRISPR-Cas9 gene editing to knock out Pi21 in Baldo, a traditional Italian rice cultivar that currently carries the functional (susceptible) allele. By introducing mutations similar to naturally occurring resistant alleles, the resulting edited plants are expected to show reduced susceptibility to P. oryzae, supporting more sustainable rice production and preserving the agronomic value of an Italian heritage variety.

Technical specifications

• Target gene: Pi21, a QTL involved in quantitative resistance to rice blast (Pyricularia oryzae).
• Editing approach: CRISPR-Cas9 with guide RNAs designed against protospacer-adjacent motifs (PAMs) located near the Proline-Rich Region of Pi21, to recreate mutations equivalent to the natural resistant allele.
• Delivery method: Agrobacterium tumefaciens-mediated transformation of scutellum-derived embryogenic calli, using a vector carrying the CRISPR-Cas9 cassette and Pi21-specific gRNA.
• Selection workflow: In vitro culture on selective medium to recover transgenic calli, followed by genotyping of T1 plants to identify edited, transgene-free segregants.
• Phenotypic validation: Evaluation of T2 plants homozygous for the edited Pi21 allele for resistance to P. oryzae.
• Prior evidence: Leaf lesion assays on the resistant cultivar Owarihatamochi, the susceptible cultivar AA, and the AA-pi21 near-isogenic line, inoculated with ten pathogen strains, showed significantly smaller lesions on AA-pi21 compared with AA, confirming the protective effect of the Pi21 loss-of-function allele.

Technology readiness level

The proposal is at an early-to-mid stage of development. The conceptual basis is supported by published evidence that Pi21 loss-of-function reduces blast susceptibility, and the host laboratory has an established rice transformation workflow for Italian cultivars. Future work focuses on generating edited Baldo plants, recovering transgene-free edited lines, and performing phenotypic validation under controlled inoculation. Additional field-level validation and regulatory steps for genome-edited crops would be required before any commercial deployment.